Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of <i>all</i>-<i>trans</i> retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of <i>de novo</i> antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (C_cc^S) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of C_cc^S to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with C_cc^S than BMS493. Interrogation of the structure of C_cc^S indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, C_cc^S self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11–15 nm in diameter. Treatment of human articular chondrocytes <i>in vitro</i> reveals that C_cc^S·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA